The present invention relates to a method of producing certain peptides containing methylated arginines that are followed by a glycine residue and that constitute immunogenic determinants of antibodies present in sera from patients with systemic lupus erythematosus, or Epstein-Barr virus and wherein the methylation is a prerequisite for reacting with said antibodies. The invention also relates to the use of said peptides for diagnosis and treatment of systemic lupus erythematosus and related diseases, diseases in which Epstein-Barr virus has been implicated.
Systemic lupus erythematosus is an autoimmune disease, in which the patient develops antibodies that react with many tissues of his own body. Dominant antibodies are directed against components of the cell nucleus, with epitopes that may be found in DNA, and in proteins that constitute small ribonucleoprotein particles (snRNPs).
The first laboratory test ever devised for this disease was the LE (lupus erythematosus) cell test. This test has to be repeated many times, before it results in a positive reaction in about 90% of the people with systemic lupus erythematosus. Also, the LE cell test is not specific for lupus, and can be positive in up to 20% of the people with rheumatoid arthritis, in some patients with other rheumatic conditions like Sjxc3x6gren""s syndrome or scleroderma, in patients with liver disease, and in persons taking drugs such as hydralazine and procainamide. The ANA test, which detects antibodies against nuclear antigens, is more specific for lupus than the LE test, and is positive in many patients that suffer from systemic lupus erythematosus. As with the LE test, a positive ANA is not diagnostic for lupus since the test may also be positive in people with scieroderma, dermatomyositis, rheumatoid arthritis, Sjxc3x6gren""s syndrome, in patients treated with certain drugs, or in patients suffering from infectious mononucleosis, liver disease, malaria etc. For these reasons and because the summed tests are expensive, new tests have been developed which are very helpful in the diagnosis of SLE. These include the anti-DNA antibody test, the anti-Sm antibody test, the anti-RNP antibody test, the anti-Ro antibody test, and tests which measure serum complement levels. Often, correct diagnosis will depend on the interpretation of many separate tests and symptoms.
The Sm antigen is a complex macromolecular structure consisting of 8 proteins (B, Bxe2x80x2, D1, D2, D3, E, F, G) associated with the U series of small RNA molecules. SmBBxe2x80x2 and SmD are considered as the major antigenic components of the complex (for review see S. O. Hoch, 1989). However, SmBBxe2x80x2 shows cross reactivity with the anti-RNP antibodies, consequently SmD is regarded as the most specific autoantigen for Sm (W. J. van Venrooij et al, 1991).
The SmD cDNA has been isolated from a human B-lymphocyte library with synthetic oligonucleotide probes, designed on the basis of the N-terminal sequence of SmD (Rokeach et al., 1988). Subsequently, it was shown that the in vitro transcription product could be immunoprecipitated by anti-Sm IgG. The D protein has since been characterized either as a doublet designated D and Dxe2x80x2 (Andersen et al., 1990) or as three polypeptides designated D1 (16 kDa), D2 (16.5 kDa) and D3 (18 kDa) (Lehmeier et al., 1990), D1 being identical with the SmD cloned by Rokeach et al.(1988). The sequence of D2 and D3 is substantially different from D1.
Over the years, several research groups have reported on the use of recombinant SmD and of SmD derived peptides and have published conflictory data. Rokeach et al. (1992a) expressed SmD1 in E. coli and in S. cerivisiae, but in contrast to the reactivity of natural SmD from HeLa cells, most of the patient anti-SmD sera bound recombinant SmD1 at a level not significantly higher than normal human sera. Nevertheless, the same group (Rokeach et al., 1992b) has performed epitope mapping based on multiple fusions between the TrpE gene and fragments of the SmD coding sequence, expressed in E. coli. Two patterns of anti-Sm reactivity emerged: discontinuous epitopes were found scattered over the full-length antigen, and a dominant epitope was located at the C-terminus, from amino acid 87 to 119 (Rokeach et al., 1992b). Using synthetic peptides, Barakat et al. (1990) showed that the N-terminus (peptide 1-20) and peptide 44-67 could be used as a valuable probe for SLE diagnosis although their results did not match the anti-SmD reactivity obtained by the traditional assay (patent EP-B-0491014). Using a similar strategy, Sabbatini et al. (1993a) have identified a dominant epitope in the C-terminal region of SmD1 (aa95-aa119) confirming the results of Rokeach et al. (1992b), but opposing the results obtained by Barakat et al. (1990). The most recent work on epitope mapping of SmD1 by means of synthetic peptides (James et al. 1994) showed that 8 of 9 SmD positive sera (precipitin positive) are reactive with the sequence spanning octapeptides 92-112. An additional epitope, clearly reactive with 7 of 9 SmD positive sera was located in the region of amino acid 82-90. Finally, a SmD-like epitope was recently identified by Rivkin et al. (1994) and consists of a (Gly-Arg)9 dipeptide repeat (homology with the C-terminus). In contrast to the SLE specificity of anti-Sm antibodies, the defined epitope is also recognized by patients with other autoimmune diseases (rheumatoid arthritis, scleroderma, Sjxc3x6gren""s syndrome). The xcex2galactosidase fusion protein in E. coli of the above mentioned epitope was reactive with 35% of the SLE sera, but only 6 out of these 32 positive sera were reactive with the native SmD protein indicating that the fusion protein is less specific than the native SmD protein. Vice versa, only four of eight SmD sera reacted with the fusion protein. It should be noted however, that SmD was also expressed as a full-size xcex2-galactosidase fusion protein in E. coli (Wagatsuma et al. 1993), but that this recombinant SmD antigen was not recognized by patient sera, although all sera recognized the natural Sm 16 kDa antigen on Western blot.
In conclusion, none of the described synthetic peptides nor the entire recombinant protein or parts of the molecule result in an immunoreactivity identical with the reactivity obtained with natural SmD.
It is an aim of the present invention to provide peptides which have a high reactivity for antibodies present in sera from patients with systemic lupus erythematosus.
Another aim of the present invention is to provide methods for obtaining said peptides.
Another aim of the present invention is to provide methods of raising antibodies specifically reactive with peptides of said peptides, thereby mimicking said peptides.
Another aim of the present invention is to provide methods of raising anti-idiotype antibodies specifically reactive with the afore mentioned antibodies.
Another aim of the present invention is to provide a pharmaceutical composition consisting of these peptides, for therapy or diagnosis.
Another aim of the present invention is to provide a diagnostic kit for systemic lupus erythematosus.
All these aims of the present invention are met by the following embodiments of the present invention.
According to its main embodiment the present invention relates to peptides containing less than 50 amino acids, comprising at least one dimer of the type XG, wherein X stands for a methylated arginine residue, and that are able to react with antibodies, with said methylation being crucial for the reaction between said peptide and said antibodies, and wherein said antibodies are present in sera from patients with systemic lupus erythematosus, or infectious, recurrent or chronic mononucleosis, or certain cancers which are related to infection with Epstein-Barr virus, such as Burkitt""s lymphoma or nasopharyngeal carcinoma.
According to a further embodiment the present invention also relates to a peptide and/or chemical structure comprising any of the above mentioned peptides, fused to a linker molecule. The present invention also relates to peptides comprising and/or consisting of tandem repeats of at least two of any of the above mentioned peptides, or branched peptides that comprises at least one of the above mentioned peptides.
According to a more specific embodiment the present invention also relates to a method for producing any of the above mentioned peptides, by classical chemical synthesis, wherein methylated arginines are substituted for unmethylated arginine residues at certain steps during the chemical synthesis. The present invention also relates to a method for producing any of the above mentioned peptides, wherein the primary amino acid sequence is produced by classical chemical synthesis, and wherein the arginine residues that precede glycine residues are subsequently methylated by contacting said peptides with a protein arginine methyltransferase. The present invention also relates to a method for producing any of the above mentioned peptides comprising the following steps: (i) transforming an appropriate cellular host with a recombinant vector in which a polynucleic acid is inserted comprising the sequence that codes for said peptide under the control of the appropriate regulatory elements such that said peptide or a protein comprising said peptide is expressed and/or secreted, (ii) culturing said transformed cellular host under conditions allowing expression of said protein or peptide and allowing a partial or optimal methylation of the arginines present in said peptide, and (iii) harvesting said peptide. The present invention also relates to a method for producing any of the above mentioned peptides comprising the following steps: (i) transforming an appropriate cellular host with a recombinant vector in which a polynucleic acid is inserted comprising the sequence that codes for said peptide under the control of the appropriate regulatory elements, such that said peptide or a protein comprising said peptide is expressed and/or secreted, (ii) culturing said transformed cellular host under conditions allowing expression of said protein or said peptide, (iii) harvesting said protein or said peptide, and (iv) methylating arginine residues of said protein or said peptide by contacting with a protein arginine methyltransferase. According to a more specific embodiment the present invention also relates to any of the above mentioned methods, wherein said host cell is a bacterial host or yeast or any other eukaryotic host cell which is preferably transformed with a recombinant baculovirus.
According to a preferred embodiment the present invention also relates to an antibody raised upon immunization with any of the above mentioned peptides, with said antibody being specifically reactive with the methylated forms of said peptide, and with said antibody being preferably a monoclonal antibody. The present invention also relates to an anti-idiotype antibody raised upon immunization with any antibody as defined above, with said anti-idiotype antibody being specifically reactive with said antibody, thereby mimicking the methylated form of any above mentioned peptide, and with said antibody being preferably a monoclonal antibody.
According to a more specific embodiment the present invention also relates to an immunotoxin molecule comprising and/or consisting of a cell recognition molecule being a peptide as defined above, or an antibody as defined above, covalently bound to a toxin molecule or active fragment thereof.
According to a further embodiment the present invention relates to any of the above mentioned peptides or antibodies or immunotoxine molecules or a composition thereof for use as a medicament. Said use can have the purpose of a medicament for treatment or of a diagnosticum for any of the following auto-immune diseases: systemic lupus erythematosus, discoid lupus erythematosus, scleroderma, dermatomyositis, rheumatoid arthritis, Sjxc3x6gren""s syndrome, or for diseases in which Epstein-Barr virus can be implicated such as Burkitt""s lymphoma or nasopharyngeal carcinoma, or infectious, recurrent or chronic mononucleosis. More specifically, the present invention relates to a treatment for auto-immune diseases by increasing the size of antigen-immune complexes, thereby improving the clearance of the formed immune complexes. The present invention also relates to a treatment for auto-immune diseases by inducing a state of systemic hyporesponsiveness to the auto-antigen after oral administration of any of the above mentioned peptides or antibodies or immunotoxine molecules or a composition thereof, thereby preventing the pathogenic production of anti-self antibodies like anti-Sm antibodies or anti-DNA antibodies. The present invention also relates to a diagnostic kit for use in detecting any of the afore mentioned diseases, wherein said kit comprises at least one of the above mentioned peptides or antibodies, and with said peptide or antibody being possibly bound to a solid support. More preferably said kit is comprising a range of said peptides or said antibodies, possibly in combination with native methylated SmD1 or SmD3 or Sm69 and recombinant unmethylated SmD1 or SmD3 or Sm69, wherein said peptides are attached to specific locations on a solid substrate. More preferably said solid support is a membrane strip and said polypeptides are coupled to the membrane in the form of parallel lines. It has to be understood that certain peptides, or antibodies as defined above, alternatively, are not attached to a solid support but are provided in the binding solution to be used as competitors and/or to block other antibodies that are present in sera from patients with autoimmune diseases other than SLE, thereby decreasing or eliminating possible cross-reaction and/or aspecific binding.